Biochemical changes in the midgut during metamorphosis in Apis cerana indica

2009·4.1 MB·

by  Deepak D Barsagade

72
Ualteres,Vol.1,No.l,2009
i
Biochemical changes in the midgut during metamorphosis in Apis
ceranaindica
Deepak D. Barsagade^ Kalpana M. Kelwadkar and Mangala N. Kadwey
DepartmentofZoology, RashtrasantTukadojiMatiarajNagpurUniversity, Nagpur-440033.
femail: [email protected])
Abstract
The digestive cells of midgut are responsible for the secretion of various enzymes
and absorption of nutrients. During the process of metamorphosis midgut passes
through, histolysis and histogenesis. As a result, destruction of larval tissue and
construction of adult tissue occurs. The present work thus carried out is to know
the changes that occurr in the biomolecules like DNA, RNA, Proteins, Carbohydrates
etc., in relation with the remodeling of gut. Along with these biomolecules, various
enzymes like amylase, invertase, protease and lipase are also estimated to know
their status during metamorphosis of midgut in Apis cerana indica.
Keywords- Apis cerana indica, Midgut, Metamorpliosis, Biomolecuies.
Introduction
Apis cerana indica is one of the in digestiveprocesses like, enzymesecretion,
indigenous species of honeybees, used for epithelial tissue regeneration, absorption of
apiculture in India. Thealimentarycanal ofthe nutrients, working of gut musculature and
bee can be divided into three distinct regions maintenanceofgutpH (Prabhuand Sreekumar,
foregut, midgutand hindgut(Snodgrass, 1935 1994 and Sunitha eta!., 1999). In Apiscerana
and 1956). The midgut is the main siteforthe indica remodeling of gut occurs through
digestive processes in most of the insects histolysis and histogenesis during
(Wigglesworth, 1972 and House, 1974), the metamorphosisbutno information isavailable
midgut epithelium is composed of columnar regarding biomoleculeconcentrationandprotein
cellswhich secretes the enzymes, carries out profile in the midgut cells during
digestionandabsorption (Wigglesworth, 1977). metamorphosis. Therefore, presentwork has
The ultrastructural and cytochemical aspect been undertaken to know the changes in
indicates that the midgut is remodeled during biomolecules, enzymes and protein profile in
metamorphosisandshowstheprogrammedcell the midgutepithelial cells oflarvae and pupae
death in the honey bee larvae and pupae ofApiscerana indica.
(Gregorc and Bowen, 1996 and 1997, Cruz-
landim and Cavalcante2002and 2003, Neves Materials and Methods
at a!., 2003 and Barsagade and Kelwadkar, Biochemical Techniques
2008). The peptidesofinsectbrain and midgut Extract Preparation:
aresuggestedtohaveimportantregulatoryrole Themidgutsweredissectedoutfromthe
BiochemicalchangesinthemidgutduringmetamorphosisinApisceranaindica 73
larval and pupal stages, in ice-cold saline/ contents of the midgut were removed by
Ringer'ssolution. The dissected midgutswere injectingthe insectsaline intotheopen midgut
homogenized using pestle and mortar for 5 tube and contents were flushed out. The
minutes at room temperature in the Ringer's epithelial tissue was washed in insect saline
solution. 25 mg tissue/ml volumes of Ringer and transferred to fresh saline. The extract of
solutionwasadded, samplewashomogenized the midgut epithelia was prepared, the
at 3000 rpm and the supernatant after homogenatehavingaconcentrationequivalent
centrifugation was used forestimation oftotal to two midgut epithelia / 10 ml saline was
concentrationofproteinaccordingtotheLowery bioassayed for its effect on in vitro digestive
et al., (1951) method. The total carbohydrate enzymesecretion in preparationofthe midgut.
wasestimated byPhenol-Sulphuricacid method The midgut preparation was incubated with 2
of Dubois et al., (1962), DNA and RNA by ml of incubation solution (midgut epithelial
Bruton's Diphenylamine and Dische-Orcinol extract)inthebioassayapparatusfor30minutes
methods of Searcy and Maclnnis (1970a and with a bubbling gentle stream ofoxygen. After
1970b). incubation, the midgut preparation was taken
out and washed in insect saline, the gut was
Biochemical Estimation of Digestive opened and contents were collected in 0.5 ml
EnzymeActivity distilled water for estimation of protease and
Preparation of enzyme extract: amylaseactivity. In controlexperiments, ligated
The enzyme extract preparation was midguttubeswere incubated in insect saline.
carried out according to the method of
Applebaum et al., (1964) with some SDS-PAGEElectrophoresis
modification. Onlymidgutwastaken from both Midgut extraction was carried out by
larvae and pupae, kept in ice-cold insect themethodofLaemmli, (1970)with someminor
Ringer's solution, accurately weighed and modifications(Barsagade, 1998).Themolecular
homogenized for 3 minutes at 0°C in ice-cold weightofthe protein bandswasestimatedwith
citrate phosphate buffer(pH-6.8) using pestle the helpofGel.Doc.
and mortar. The midgutwassuspended in ice-
coldbufferand madeupto1 ml,thehomogenate Results
was centrifuged at 10,000 rpm for 15 minutes BiochemicalAnalysis
and the supernatant was used as an enzyme Concentration of the biomolecules
for estimation ofdigestiveenzymeactivity.The present in the midgut epithelial cells of larvae
enzymeactivityofamylaseand invertasewere and pupaewerefoundas-
estimated by the methods of Ishaaya and
Swirsky(1970), whileproteaseactivitybySnell Total DNAconcentration
and Snell (1971) method and lipaseactivity by The total midgut DNA concentration in
the method ofCherryand Crandel (1932). fifth instar was about 0.58 ± 0.08 pg/mg, it
decreased to 0.3 ± 0.06pg/mg in early pupa.
Preparation of midgut for bioassay ThetotalDNAconcentrationthereafterincreased
experiment: gradually up to 0.45 ± 0.04 pg/mg and 0.66 ±
Alimentary canal was dissected out in 0.08 pg/mg in mid pupa and late pupa
insect saline, the midgut was separated. respectively(Fig.1).
Halteres,Vol.1.No.1,2009 74
Total RNAconcentration Total Carbohydrate concentration
The total RNA concentration in the fifth The total carbohydrate concentration in
instarlarvaewas estimated as 12.2 ± 0.99 |jg/ the fifth instar larvae was estimated as 0.70 ±
mgwhile itwas7.12±0.59|jg/mg inearlypupa, 0.0046 pg/mg while it was 0.67 ± 0.0023 pg/
increased to 9.12 ± 1.01 pg/mg in mid pupa mg in early pupa, increased to 0.68 ± 0.0021
and was found to be 18.6 ± 1.01 pg/mg in the pg/mg in mid pupaandwasfoundto be0.72 ±
late pupal stage (Fig-2). 0.0041pg/mg in the late pupal stage (Fig-4).
Total Protein concentration Midgut Digestive Enzyme
Thetotal midgutproteinconcentration in The digestive enzymes, amylase,
fifth instar was about 4.5 ± 0.29 pg/mg. It invertase, protease, and lipase in the midgut
decreased to 2.04 ± 0.22pg/mg in early pupa, have been demonstrated qualitatively and
the total protein concentration thereafter quantitatively. Enzyme estimation in the larval
increased gradually up to 3.73 ± 0.31 pg/mg and pupalstages issummarized intable 1. and
and6.5±0.14 pg/mg in mid pupaand latepupa fig.5.
respectively(Fig-3).
Table-1: Enzyme estimation in larval and pupal stages of Apis cerana indica. (Mean ± SD)
Enzyme V-instar Early Pupa Mid Pupa Late Pupa
S. No. Mg/mg Mg/mg pg/mg pg/mg
Amylase 0.19± 0.0054 0.53± 0.0187 0.028± 0.022
1. 0.32± 0.014
2. Invertase 0.34± 0.015 0.44± 0.0122 0.77± 0.015 0.80± 0.055
3. Protease 0.75± 0.0057 0.78± 0.01 0.43± 0.0291 0.82± 0.021
4. Lipase 4.5± 0.05 5.5± 0.291 2.9± 0.212 3.2± 0.254
Effect of Midgut Extract
Bioassayexperimentwas conducted to midgut /minute after the incubation in midgut
studytheeffectofmidgutextractonthe midgut extract for 30 minutes while, it was observed
amylaseand proteaseactivity inthefifth instar 0.51 ± 0.071 mg glucose/midgut/minute in the
larvaeofhoney beeApisceranaindica(Fig.6). normalcondition.
During the bioassay experiment the midgut
extractshowedthesignificant(P<0.0001)effect Proteaseactivity:
elevating amylaseand proteaseactivity infifth The midgut protease activity was
instar. measured about 0.94 ± 0.091 mg protein/
midgut/minuteafterincubation in midgutextract
Amylaseactivity: for30 minuteswhile, itwas noticed about0.79
The midgut amylase activity was ± 0.083 mg protein/midgut/minute in normal
measured about 0.71 ± 0.089 mg glucose/ condition.
BiochemicalchangesinthemidgutduringmetamorphosisinApisceranaIndica 75
ElectrophoreticAnalysis weightinfirsttofifth instarlarvae, early, mid and
TheSDS PAGEelectrophoreticanalysis late pupae of Apis cerana indica .While an
of midgut extract demonstrated about eleven additionalbandof12KDproteinwasfoundonlyin
bands of protein among which six bands were late pupa (Fig. 7).
predominantrangingfrom26-65kDamolecular
0.8 20 -
.E 18 -
o 16 -
1 14 -
1 o»12^ rir
<o a. 8
0
\
V EP MP LP
Developmentalstages DevelopmentalStages
Fig.1 TotalDNAconcentrationduring Fig.2 TotalRNAconcentrationduring
post-embryonicdevelopment post-embryonicdevelopment
o>7
E
0.74
•S5
0.72
o
14 1-
5 o. 0.7
" E
i
oo 2 0.66 i n
I' •e 0.64
o
0.62
EP MP MP LP EP
DevelopmentalStages DevelopmentalStages
Fig.3 Totalproteinconcentrationduring Fig.4 Totalcarbohydrateconcentration
post-embryonicdevelopment. duringpost-embryonicdevelopment.
Abbreviations V -Vth Larval stages EP- Early pupa
: ; ;
fAP - Mid pupa LP -Late pupa
;
Halteres,Vol.1,No.1,2009
eContoliExperimental!
in
ntration
r
Seo.6h illH
u :
co s
s ftp!
Enzyme liHii
1
.3
Amylase Pratesae
Enzymes
Fig.5 Estimationofenzymesinlarvalandpupal Fig.6 Effectofmidgutextractonmidgutactivity
stagesduringpost-embrvonicdevelopment. in V-instarlarva.
81.OCX)
65.000
43.OO0
29.000
21.100
14.300
6.500
3,000
KO MARKER Ml IV VI VII Vlli
PMW-L
Fig.7 SDS-PAGE analysis of the midgut extract of larvae ofApis ceratm indica
showingthepresenceofproteinpattern.
Abbreviation; 1- first instar. 11- second instar, III - third tnstar. IV - fourth instarand V- fifth
instar.Vl-eartypupa,Vil-midpupa,VIIMatepupa.
BiochemicalchangesinthemidgutduringmetamorphosisinApfsceranaindica 77
Discussion activity (Horie, 1970, Banergee and Saxena,
Duringthepostembryonicdevelopment, 1983, Wajiro et ai, 1984 and Zufelato et ai,
the differentiation of midgut epithelial cells 2004). The higher level of enzyme acid
occurs in honey bee (Chapman, 1985a and phasphatase activity in the larval peritrophic
1985bandCruz-landim andCavalcante, 2003) membrane in Apismeilifera is mostly used for
while in the successive development, digestion (Cavalcanteand Cruz-landim, 2004).
replacementoflarval midgutepithelial cellsto Similarly activityofotherfourteenenzymeswas
adultbyformation ofnewepithelium isfound in determined in the midgutoiApismeiliferaand
Apisceranaindica(Barsagadeand Kelwadkar, these enzymes were alfa-amylase, 4 alfa-
2008). In the last larval instar the midgut glucosidase, 3 protease, 5 amino peptidase,
epithelial cells, produce various digestive lipase etc. (Banerjee and Sexena,1983).
enzymesthathelpforthefooddigestion inApis According to Mahmoud (2007), the high food
meilifera(CavalcanteandCruz-Landim,2004). metabolism level depends on the protein
During larval development midgut cells are concentration in themidgutand haemolymph,
replaced by the other cells depending upon it may reflect the digestion level with acid
death of larval digestive cells, proliferation of phasphataseandotherdigestiveenzymes. The
regenerativecellsoccursin ordertoconstitute present investigation reveals that the
a new digestive epithelium in the adult. Apis concentration ofbiomolecules viz. total DNA,
meilifera (Geogorc and Bowen, 1997) and in total RNA, totalproteinandtotalcarbohydrates
Apisceranaindica, (Barsagadeand Kelwadkar, in the fifth instar larvae ofApis cerana indica,
2008). shows initial depletion and then a significant
The midgut columnar epithelial cells rise because of involvement of total protein
containingthebiomoleculessuchasDNA, RNA consumption in the midgut for synthesis of
and Proteinsareactivelyengaged intheprotein digestiveenzymes.
synthesis in orderto secure various digestive The study also supports the findings of
enzymes during the larval-pupal earlierworks, showing theamylase, invertase,
metamorphosis(Fuji, 1979,Wigglesworth, 1972 proteases and lipaseactivityduring the larval-
andChapman, 1998). pupal metamorphosis in Apis cerana indica.
In Apis cerana indica, total DNA, total CavalcanteandCruz-landim (2004), noticedthe
RNA,totalproteinsandtotalcarbohydrateswere mass range ofdifferent proteins varying form
intensely demonstrated in the nuclei and 19to 142 kDa, increasedgreatlyfrom larvaeto
perikaryaofcolumnarepithelialcells, asfound pupaeandtendstodecreaseduring pupation,
in Apisfloreaand Apismeilifera, described by until the phase of brown eye pupae and
earlier workers showing their role in protein increased again in the pupae with pigmented
synthesis (Pearse, 1968; Ban and Prezlec, body (pharateadult). InApisceranaindica, 11
1974). The similar role ofthese biomolecules bandswereobserved, amongwhich six bands
may be played in the protein synthesis during were predominant ranging from 26-65 kDa,
metamorphosis in Apisceranaindica. found inthemidgutextractofdeveloping larvae
Various biochemical tests reveal the and pupae, supportingtheearlierstudies.
presenceofamylase, invertase, proteaseand
lipase activity in the midgut of larvae of Apis
cerana indica which are very specific in their
.
Halteres,Vol.1.No.1.2009 78
References serum lipase activity. Annual Journal of
Applebaum,S.W.,Harper, I.andBondi,A. 1964.Amylase Physiology 100: 266.
secretion in the larvae of Prodenica litura F.
Insecta. Comparative biochemistry and Cruz-landim, C. andCavalcante, V. M.2002. Cellnucleus
Physiology 13: 107-111. activity during post-embryonic development of
ApismelliferaL.(Hymenoptera:Apidae).Genetic
Ban, A. and Przelec 1974. The influence of starvation and Molecular research 1(2): 131-138.
on ribonucleic acid and protein synthesis in
midgutepitheliumofGallariamellonellalarva. Cruz-landim,C.andCavalcante,V.M.2003.Ultrastructural
Folia Histochemica et Cytochemica 11(314): andCytochemicalaspectsofmetamorphosisin
177-183. the midgut ofApis mellifera L. (Hymenoptera:
Apidae : Apinae). Zoological Science 20(9):
Banergee, A. and Saxena, P. 1983. Digestive enzymes 1099-1107.
inthealimentarycanalandassociatedglands
of foragers ofApis cerana indica. Lucknow: Dubois,M.,Giller,K.A.,Hamilton,J.K.,Rebers,P.A. and
DepartmentofZoology,UniversityofLucknow. Smith, F. 1956. Colorometric method for
determination of sugar and related
Barsagade, D.D. 1998. Studies on the silkgland substances. Analytical Chemistry 28:350.
development and silk protein syntheisis in
tasar silkworm, Antheraea mylitta(D). Ph.D. Fuji, H. 1979. Studies on function and structural
thesis, Nagpur University, Nagpur, India. conversion ofthe midgutduring larval, pupal,
transition in silkworm, Bombyx mori (4).
Barsagade, D.D. and Kelwadkar, M. K. 2008. Hormonal regulation of metamorphosis.
Ultrastructural changes in themidgutduring Science Bulletin of Faculty of Agriculture
metamorphosis in Apis cerana indica. Kuyushu, University 33 (2-3): 127-166.
Journal of Industrial Entomology 6{1): 29-
35. Gregorc, A. and Bowen, I .D. 1996. Scanning electron
microscopyofthehoneybee(ApismelliferaL.)
Cavalcante, V.M. and Cruz-landim, C. 2004. larvae midgut. ZbVet. fAk.-Univ.Ljubl. 33: 261-
Electrophoretic protein pattern and acid 269.
phosphates activity in the midgut extract of
ApismelliferaL. (Hymenoptera:Apidae)during Gregorc,A.andBowen,I.D.1997.Programmedcelldeath
metamorphosis. Neotropical Entomology 33 in the honey-bee (Apis mellifera L.) larvae
(2):169 -172. midgut. Cell Biology International 21(3): 151-
158.
Chapman, R.E. 1985a. Co-ordination of digestion, in:
Kerkut, G.A. and Gilbert, L.I. (eds.). Horie, K. 1970. Physiological studies on the digestive
Comprehensive Insect Physiology, tract oflarvae ofthe Silkwomri, Bombyx mori
Biochemistry and Pharmocology. Pregaman L. II Carbohydrates in the digestivejuice and
Press, Oxford 4: 213-240. midgut. BulletinofComparativeSericulture15:
365-380.
Chapman, R.E. 1985b. Co-ordination of digestion. In:
Kerkut, G.A. and Gilbert, L.I. (eds.). House, H. L. 1974. PhysiologyofInsecta. In: Rockstein,
Comprehensive Insect Physiology, M. (ed.). International Conference on
Biochemistry and Pharmocology. Oxford, Apiculture in Tropical Climates, Division
Pregaman Press, 4:165-211. EntomologylARI,NewDelhi:367-371
Chapman, R.E. 1998. Endocrinesystem,Atextbookof Ilshaaya, I. andSwirsky, E. 1970. Invertaseand amylase
insect structure and function IV. Cambridge: activity in armored scales Chrysomphalus
Cambridge University Press. aoridamandAonidiellaauranti.Journalof Insect
Physiology 16 1599-1606.
:
Cherry, I. S. and Crandell, L.A. 1932. Determination of
Biochemicalchangesinthemidgutduringmetamorphosisin4p/sceranaIndica 79
Laemmli, U.K. 1970. Cleavageofstructuralproteinduring BiochemicalJournal62: 315-323.
the assembly of head of bacteriophage T^.
Nature227: 680.85. Snell, RD. and Snell. C.T. 1971. Colorimetricmethodof
analysis. New York: 3'*^ Edition Academic
Lowry, L. H., Rosebrough, N.J., FarrA.L., andRandall. R. Press.
J. 1951. Proteinmeasurementwithfolin-phenol
reagent. Journal ofBiologicalChemistry2193 : Snodgrass, R.E. 1935. Principle of insect morphology.
65-75. New York: New York Press.
Mahmoud, E. Z. 2007. Factor affecting on the food Snodgrass, R. E. 1956.Anatomyofhoneybee. NewYori<:
metabolism insomehoneybee races. Journal Comell UniversityPress.
ofApplied sciences Research 3(4): 311-316.
Sunitha, V.B.. Reena, T, Harshini. S. and Sreekumar, S.
Neves, C.A., Gitirana, L. B. and Serrao, J. E. 2003. Ultra 1999. IsgutpH regulated bymidgutendocrine
Structure, of the midgut endocrine cells in system. IndianJoumalofExperimental Biology
Melipona quadrifascita anthidioides 37: 423-426.
(Hymenoptera: Apidae). Brazilian Journal of
Biology63: (4). Wajiro,M., Fuji, H. andSakaguchi, B. 1984.Activityand
polymorphism of digestive juice amylase in
Pearse,A.G1968.Histochemistry-TheoriticalandApplied. various strains of Bombyx Mori. Journal of
London: J. andA. Churchill Ltd. SericultureSciencesofJapan55(6):496-500.
Prabhu, V. K. K. and Sreekumar, S. 1994. Endocrine Wigglesworth, V. B. 1972. The principles of insect
regulationoffeedinganddigestionininsects.In: physiology 7"' Ed. NewYoric: N. Y. John Wiley
Agrawal, P. (ed.). Entomological Research, and Sons.
Scientific Publishers, Jodhpur: 117-135.
Wigglesworth, V. B. 1977. The principles of insect
Roth,T. F. and Porter, K. R. 1964.Yolkproteinuptakein physiology, London: Methuen Press.
oocyte of mosquitoAedes aegypti L. Journal
ofCell Biology20: 312-332. Zufelato. M. and Laureno, A. P., Simoes Z. L., Jorge,
J.A.,Bitondi,M.M.2004.Phenoloxkiaseactivity
Searcy. D.G. and Maclnnis, A. J. 1970a. Biochemical in Apis mellifera honey bee pupae, and
estimation ofDNAbyBurton's Diphenyamine ecdysteroid dependent expression of the
method. BiochemicalJournal62: 215-223. prophenoloxidase m-RNA. Insect
Biochemistry and Molecular Biology 34(12):
Searcy, D.G. and Maclnnis, A. J. 1970b. Biochemical 125-68.
estimationofRNAbyDische-Orcinolmethod.